The blood clotting cascade is activated when there is a change in the integrity of the blood circulatory system which results in the escape of blood. Generally, circulating platelet collect at the site of the wound, and upon exposure to air, the platelets rupture releasing the serine protease thromboplastin (Factor III). In the presence of vitamin K, Ca.sup.++ ions and several other essential factors, thromboplastin catalyzes the conversion of prothrombin (Factor II) to thrombin, a serine protease with a molecular weight ranging from 36 kDa to 37 kDa. The newly formed thrombin catalyzes the hydrolysis of fibrinogen to form fibrin strands. The resulting fibrin in combination with activated platelets form a plug or clot to seal the wound, and contraction of the clot pulls the wound together preventing further bleeding (Clegg et al., Advanced biology principle and applications. London: John Murray Publishers, Ltd. (1994); Wallace et al., Biology: The science of life. Scott, Foresman and Company (1986)).
Thrombin can be formed from prothrombin in vitro by three known processes. One process involves incubating prothrombin with isolated thromboplastin in the presence of calcium chloride. This process is described, for example, in EP 0439156A. The disadvantage of using this method is that the thromboplastin typically employed is extracted from bovine lungs. The extraction process is time consuming and the resulting bovine thromboplastin can be contaminated with viruses or bacteria which carries the risk of transmitting infectious diseases. In addition, trace amounts of bovine thromboplastin that might be present in the resulting purified thrombin can cause immunological reactions in the host receiving thrombin produced by this method.
Another in vitro process for converting prothrombin to thrombin involves treating a mixture of prothrombin, Factor Xa, Factor Va and phospholipids with calcium ions. This process is described, for example, in U.S. Pat. Nos. 5,219,995 and 5,907,032. According to U.S. Pat. No. 5,907,032, the prothrombin mixture may also contain additional clotting factors, such as Factor X, Factor V, Factor IX, Factor IXa and trace amounts of thrombin. Thrombin produced in this manner has a specific activity ranging from about 250-700 U/mg. The requirement for additional clotting factors and the low specific activity are undesirable features of this process.
Similarly, in other processes, purified prothrombin is treated with activated Factor X, Factor V, phospholipid and calcium ions under physiological conditions to yield thrombin, or purified prothrombin is treated with 25% sodium citrate in the presence of coagulation factors such as Factor VIII, Factor IX and Factor X (Seegers, W. H., Proc. Soc. Exp. Biol. Med., 72:677-80, 1949; Teng et al., Thrombosis Research, 22:203-12, 1981). The authors suggest that one or more of the various coagulating factors play a vital role in the process. Again, the requirement for additional clotting factors renders this process undesirable and inconvenient.
Still another in vitro process requires the use of snake venom to convert prothrombin to thrombin (Denson et al. Toxicon, 7(1):5-11, 1969; Masci et al. Biochem. Int., 17(5):825-35, 1988; Rosing et al. Toxicon, 30(12):1515-27, 1992; Stocker et al. Toxicon, 32(10):1227-36, 1994). Snake venoms are classified into four groups based on their structure and function. Group I venoms convert prothrombin to an intermediate meizothrombin. Group II and III venoms cleave peptide bonds in prothrombin to produce thrombin. However, both Group II and III venoms require additional factor such as phospholipids, Factor Va and CaCl.sub.2. Group IV venoms convert prothrombin to an inactive form of thrombin (Rosing et al., Toxicon, 30(12):1515-27, 1992). The disadvantages to using snake venom are 1) it is difficult to obtain snake venom in large quantities, 2) some forms of snake venom convert prothrombin into enzymatically inactive forms of thrombin, and 3) traces of snake venom in thrombin preparation can be extremely harmful to the patient.
As discussed above, thrombin is an essential component of blood coagulation that acts during the final stages of the blood clotting process. Due to this function, thrombin is used clinically as a styptic agent. Additionally, thrombin functions as an activator of protein C, Factor V, platelets and proteins of the complement system. Thus, it is desirable to develop processes whereby thrombin can be easily produced and readily available. However, until now, there have been no commercially viable methods to efficiently and simply produce thrombin from pure prothrombin. Moreover, there have been no methods whereby workers could convert prothrombin to thrombin in the absence of coagulating factors or other activating enzymes. Accordingly, there is a need in the art for an efficient and simple non-enzymatic method for producing thrombin from pure prothrombin in the absence of additional coagulation factors.